April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Breaking Down Barriers to AAV Mediated Retinal Transduction Through Intravitreal Injections
Author Affiliations & Notes
  • D. Dalkara
    HWNI and CChem,
    UCB, Berkeley, California
  • K. D. Kolstad
    MCB,
    UCB, Berkeley, California
  • M. Visel
    HWNI,
    UCB, Berkeley, California
  • R. R. Klimczak
    MCB,
    UCB, Berkeley, California
  • D. V. Schaffer
    HWNI and CChem,
    UCB, Berkeley, California
  • J. G. Flannery
    HWNI and MCB,
    UCB, Berkeley, California
  • Footnotes
    Commercial Relationships  D. Dalkara, None; K.D. Kolstad, None; M. Visel, None; R.R. Klimczak, None; D.V. Schaffer, None; J.G. Flannery, None.
  • Footnotes
    Support  5R21EY016994-02
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 3008. doi:
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    • Get Citation

      D. Dalkara, K. D. Kolstad, M. Visel, R. R. Klimczak, D. V. Schaffer, J. G. Flannery; Breaking Down Barriers to AAV Mediated Retinal Transduction Through Intravitreal Injections. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3008.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Adeno-associated viral gene therapy has shown great promise in treating inherited and acquired retinal disorders, with three clinical trials in progress this year. Currently, therapies aimed at gene replacement in the RPE and photoreceptors require creation of a subretinal detachment ‘bleb’ for delivery of viral vectors to these cells. The subretinal route of administration limits the viral transduction profile to the region of injection (diameter of the bleb) and compromises the retinal integrity by detachment. The purpose of this study is to understand the barriers to AAV-mediated retinal transduction via intravitreal injection and develop non-invasive ways of overcoming the barriers to transduction. To this end, we used a proteolytic enzyme to evaluate the contribution of the inner limiting membrane (ILM) to restricting AAV diffusion into the retina, particularly to the outer nuclear layer and RPE.

Methods: : AAV serotypes 1, 2, 5, 8 and 9 were produced via the triple transfection method. Each serotype contained cDNA encoding GFP driven by the ubiquitous chicken beta actin (CBA) promoter. Purified, high titer AAV (10E12-10E13 vg/ml) was injected intravitreally (5µl) into normal rats at p30. In parallel studies, a low dose of a proteolytic enzyme was co-injected with high titer AAV to mildly digest the inner limiting membrane. The safety of this treatment was assessed by ERG and visual cortex cell population responses. The resulting gene expression patterns were analyzed by imaging GFP.

Results: : AAV2 is the only AAV serotype that generated significant expression in the inner retina without enzyme treatment. Eyes treated with the proteolytic enzyme showed significant improvement in viral transduction in the outer retina for all serotypes studied, suggesting that mild enzymatic digestion of the ILM opens up new receptor binding sites that were previously unavailable to these serotypes. AAV5 showed the best transduction profile, penetrating into the outer retina and yielding GFP expression up to the RPE. ERG and cortical recordings suggest that enzymatic digestion of the ILM does not disrupt visual information processing.

Conclusions: : We show here, that using AAV vectors, transduction of the outer retina can be achieved from the vitreous. This will have important implications in treatment of retinal degenerative diseases using AAV mediated gene therapy where it is particularly important to avoid trauma and gliosis associated with subretinal injections.

Keywords: injection • proteolysis • retina 
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